Yeast Two-Hybrid Vector Library for Deubiquitinating Enzyme Screening
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Solution Overview
Problem
Current methods for identifying deubiquitinating enzymes that bind to target proteins are costly and inefficient, hindering the research into their roles in intracellular signal pathways and potential therapeutic applications, particularly for cancer treatment.
Innovation Solution
A vector library for yeast two-hybrid screening is developed, comprising vectors with genes encoding deubiquitinating enzymes such as USP1, USP7, and USP49, allowing for efficient identification of enzyme-target protein interactions, and a method for screening anti-cancer agents targeting these enzymes by measuring apoptosis induction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional methods are used to identify deubiquitinating enzymes that bind to target proteins, then identification accuracy can be maintained, but research costs increase and screening efficiency decreases
Solution Approach 1:
The patent introduces a yeast two-hybrid system as an intermediary platform to screen deubiquitinating enzymes. The system uses yeast cells as a living platform where target proteins and deubiquitinating enzymes interact, allowing high-throughput screening without requiring expensive conventional biochemical assays for each interaction test
Solution Approach 2:
The patent creates a library of deubiquitinating enzyme genes cloned into expression vectors, which are then transformed into yeast cells. This allows multiple copies of enzyme genes to be screened simultaneously in parallel yeast cultures, dramatically increasing screening throughput while reducing per-sample costs
2Reliability
If comprehensive deubiquitinating enzyme screening is performed to identify all binding enzymes, then interaction identification completeness improves, but screening complexity and time consumption increase
Solution Approach 1:
The patent segments the comprehensive screening task into manageable parts by organizing deubiquitinating enzyme genes into a structured library format. Each enzyme gene is cloned into separate expression vectors that can be individually transformed into yeast, allowing systematic screening of multiple enzymes without overwhelming complexity
Solution Approach 2:
The patent develops a universal yeast two-hybrid screening platform that can screen any deubiquitinating enzyme against any target protein. The expression vectors and screening protocol are designed to be universally applicable across different enzyme and protein targets, eliminating the need to develop separate screening systems for each combination
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The vector library enables cost-effective identification of deubiquitinating enzymes binding to target proteins, specifically USP1, USP7, and USP49, which bind to apoptosis-associated proteins like Bax, facilitating the development of anti-cancer agents by regulating proteasome-independent pathways.
Implementation Method 1
vector library for yeast two hybrid screening of a deubiquitinating enzyme that binds to a target protein
Implementation Method 2
A deubiquitinating enzyme cleaves ubiquitins bound to a target protein, thereby inhibiting the degradation by proteasomes
Data Source
AI summary
Provided is a vector library for yeast two-hybrid screening of a deubiquitinating enzyme that binds to a target protein and a method for identifying a deubiquitinating enzyme binding to a target protein using the same. Further provided is a method for screening an agent having anti-cancer activity targeting the deubiquitinating enzyme USP1, USP7, USP12, or USP49 identified by the identifying method.